HLA forms part of the Major Histocompatibility Complex located on chromosome 6. It comprises two classes - Class I antigens (HLA-A, B, C) expressed on all nucleated cells and present antigens to cytotoxic T cells, while Class II antigens (HLA-DR, DQ, DP) are expressed on antigen presenting cells and present antigens to helper T cells. Molecular typing techniques like PCR and sequence-based typing provide high resolution results for HLA typing without requiring viable cells.

2. HLA = Human Leucocyte Antigen
system
• HLA forms part of the Major
Histocompatibility Complex (MHC)
• Found on the short arm of chromosome 6
• MHC antigens are integral to the normal
functioning of the immune response.
• Essential role of HLA antigens lies in the
control of self recognition and thus
defence against micro-organisms and
surveillance.

3. Interesting facts
 HLA comprises two classes:
Class I
Class II
 Class I A,B,C most significant (other
loci eg E,F,G,H etc are not so important
in transplantation)
 Expressed on most nucleated cells
 Have soluble form in plasma
 Are adsorbed onto platelets (some
antigens more readily than others)

4. More interesting facts
• Erythrocytes will adsorb some Class I
antigens viz. blood group system (B7,A28,
B57….)
• HLA B most polymorphic system and
studies have shown is most significant
followed by A and then C
• 45Kd glycoprotein comprising three heavy
chain domains, non-covalently associated

5. More interesting facts
• with beta-2-microglobulin (coded
chromosome 15) which plays an important
role in the structural support of the heavy
chains.
• Class I molecules are assembled within the
cell and ultimately sit on the cell surface
with a section inserted into the lipid bilayer
of the cell membrane and a short
cytoplasmic tail where they present antigen
in the form of peptide to cytotoxic T (CD8+)
cells

6. More interesting facts
• HLA Class II five loci DR, DQ, DP, DM and
DO
• HLA DR, DQ, DP most significant
• Expressed on B lymphocytes, activated T
lymphocytes, macrophages, endothelial
cells i.e. immune competent cells.
• Comprise 2 chains encoded by HLA genes,
alpha and beta each with 2 domains.
• Hypervariable region is in the beta 1 domain

7. • More interesting facts
 HLA Class II present peptide in the cleft to
helper T (CD4+) cells. Thus Class II
presentation involves the helper-function
of setting up a general immune reaction
involving cytokine, cellular and humoral
defence.
 The role of Class II in initiating a general
immune response is why they only need to
be present on immunologically active cells.

8. Major Histocompatibility Complex
(MHC)
• The MHC is a closely linked complex of genes
that govern production of the major
histocompatibility
• In humans, MHC resides on the short arm of
chromosome 6
• Three genes (HLA-A, HLA-B, HLA-C) code for
the class I MHC proteins
• Several HLA-D loci determine the class II MHC
proteins i.e. DP, DQ and DR
• HLA genes are very diverse (polymorphic)
i.e. there are many alleles of the class I and II
genes

9. • Major Histocompatibility Complex
(MHC)
It produces cell surface markers:
1. Self from non self identification.
2. Immune regulation.
3. Immune responsiveness.
4. Disease association of resistance.

10. Major Histocompatibility Complex
(MHC)
 Between the class I and class II gene loci,
there is a third locus (Class III)
 This locus contains genes encoding tumor
necrosis factor, lymphotoxin and two
complement components (C2 and C4)
 Class III antigens do not participate in MHC
restriction or graft rejection

11. MHC Genes

12. • MHC Class I Antigens
 Class I MHC antigens are : HLA-A, HLA-B
and HLA-C
 These antigens are glycoproteins found on
surfaces of all nucleotide human cells and
on platelets
 HLA-A contains 24 different antigenic
specificities,
HLA-B contains 52 and HLA-C contains 11
 Class I MHC antigens are involved of MHC
restriction of cell mediated cytotoxicity

13. • MHC Restriction
 Endogenously processed cytosolic
peptides in virus infected cells or
tumor cells are transported to the
surface of the cells.
 They bind to MHC I molecules to be
recognized by cytotoxic T-cells which
then kill these cells.
 In other words;
T-cells are only activated when they
recognize both antigen and class I MHC
molecules in association.

14. MHC Class II Antigens
• Class II antigens are: HLA-DP, HLA-DQ, HLA-
DR antigens.
• These antigens are glycoproteins found on
the surface of macrophages, B-cells,
Dentritic cells, langerhans cells of skin and
activated T cells.
• HLA-DP contain 6 different antigenic
specificities, HLA-DQ contains 9 and HLA-
DR contains 20.

15. • .
No rearrangements or somatic changes
Diversity is derived from 1) Gene families
2) Genetic polymorphism

16. MHC Class II Antigens
• Helper T-cells recognize antigens on
antigen-presenting cells only when the
antigens are presented on the surface of
cells in association with class II MHC.
• Class II antigens react with the CD4
molecule on the helper T-cells which
secrete cytokines.

17. Class I MHC and Class II
MHC
MHC Class I MHC Class II
Nomenclature HLA-A, HLA-B, HLA-C HLA-DP, HLA-DQ,
HLA-DR
Found on All nucleated somatic
cells
Macrophages, B-cells,
Dentritic cells,
langerhans cells of
skin and activated T
cells
Recognized by CD8 TC cells CD4 TH cells
Functions Presentation of Ag to
TC cells leading to
elimination of tumor or
infected host cell
Presentation of Ag to
TH cells which secrete
cytokines

18. TYPING METHODS
• SEROLOGY used to be the ‘gold’
standard. Now being superseded by
molecular techniques as they become
more robust and time efficient.
• CELLULAR rarely used now. Originally
used for Class II typing.
• MOLECULAR fast becoming the method of
choice. Many laboratories test of choice.

19. SEROLOGY
• Complement Dependent Cytotoxicity
(CDC)
• Viable peripheral blood lymphocytes
are obtained by discontinuous density
gradient centrifugation using Ficoll /
Tryosil or Ficoll / Sodium Metrizoate at
a density of 1.077 at 19º - 22ºC.
• Microlymphocytotoxic test: 3 stages

20. Microlymphocytotoxic test
• 1.Viable lymphocytes are incubated
with HLA specific antibodies. If the
specific antigen is present on the cell
the antibody is bound.
• 2.Rabbit serum as a source of
complement is added, incubate. If
antibody is bound to the HLA antigen
on the cell surface it activates the
complement which damages the cell
membrane making it permeable to
vital stains.

21. Microlymphocytotoxic test Contd….
• 3.Results are visualised by adding dye
usually a fluorochrome eg Ethidium
Bromide although both Trypan Blue and
Eosin have been used in the past.
• If the reaction has taken place the EB
enters the cell and binds to the DNA.
• For ease double staining is normally used.
We use a cocktail of Ethidium Bromide and
Acridine Orange, quenched using Bovine
Haemoglobin to allow simultaneous
visualisation of both living and dead cells.

22. Microlymphocytotoxic test Contd….
• Test is left for 10 minutes and then read
using an inverted fluorescent microscope.
• A mixture of T and B lymphocytes can be
used for HLA Class I typing.
• B lymphocytes are required for HLA Class II
typing by serology. (Normal population 85-
90% T and 10-15% B cells)
• This can be achieved using a number of
methods.

23. Microlymphocytotoxic test Contd….
• In the past neuraminidase treated sheep red
blood cell rosetting and nylon wool have
been used.
• Immunomagnetic bead separation is the
current method of choice.
• It utilises polystyrene microspheres with a
magnetisable core coated in monoclonal
antibody for a HLA Class II b chain
monomorphic epitope. Positive selection.

24. Pros and cons
• Pros:
• Easily performed does not require expensive
equipment.
• Takes around three hours to perform
• Low level resolution, with good antisera reliable
results
• Cons:
• Requires large volumes of blood
• Requires viable lymphocytes
• Difficult to find good antisera for rarer antigens in

25. Cellular typing
• Not / Rarely used by laboratories
these days.
• Requires panels of homozygous
typing cells.
• Cell culture method therefore takes a
long time. Labour intensive involves
use of radioisotopes.

26. Molecular typing
• All commonly used molecular methods
require good quality genomic DNA. There
are numerous methods for extraction of
DNA from whole blood.
• There are ‘in house’ methods based on
Miller et al’s Salting Out which are cheap
and easy but labour intensive.
• There are also numerous commercial kits
available such as individual matrix capture
columns, beads and semi automated
systems. This however can increase the
cost per extraction from around 65p to
£3.60p.

27. Molecular typing Contd…….
• All methods rely on DNA extraction from the
nucleated cells following cell lysis and
protein digestion.
• The application of molecular techniques to
HLA typing began around 1987 when the
Southern Blot technique was used to
identify restriction fragment length
polymorphisms (RFLP’s) associated with
known serological DR/DQ and cellular Dw
defined specificities.
• Around 1992 polymerase chain reaction
(PCR) methods were developed.

28. Molecular typing Contd…….
• PCR
• Three steps per cycle– denaturation,
annealing and extension.
Amplification is exponential yielding
2 power n where n = number of
cycles.
• The introduction of the
programmable Thermal Cycler
revolutionised the use of PCR within
the routine laboratory.

29. Molecular typing Contd…….
• PCT SSP (Sequence Specific Priming)
• Can be used for HLA Class I and II typing
using a panel of primer pairs either for low
to medium resolution whereby primers
amplify groups of alleles or high resolution
whereby primer pairs amplify specific
alleles. Each PCR reaction takes place in a
separate tube therefore the number of
tubes depends on the level of resolution.
Each tube also contains a pair of primers
for part of the human growth hormone gene
as an internal control. These are at a much
lower concentration thus do not compete
with specific primers.

30. Molecular typing Contd…….
• Electrophoresis is used following
amplification. PCR product is run out on an
agarose gel containing ethidium bromide.
Each product moves according to its size
and is compared to a molecular weight
marker.
• Interpretation: every tube should produce
an identical sized product as internal
control and either a specific band or not
dependent on whether the allele(s) is/are
present or not.
• Results are visualised using 312nm UV
transillumination and recorded either by
video imaging or polaroid photography.

31. Molecular typing Contd…….
• PCR SSOP ( Sequence Specific
Oligonucleotide Probes).
• ‘Dot blot’ in house method usually whereby
one labels ones own probes with
Digoxigenin.
• ‘Reverse dot blot’ normally commercial
where specific oligonucleotide probes are
attached to a nylon membrane. Dynal and
Innotrans for example produce such kits.

32. Molecular typing Contd…….
• Amplification: DNA of interest is amplified by
a single pair of biotinylated primers which
flank the whole of exon eg exon 2 of the HLA
DRB1 gene. PCR amplifies all the alleles in
the exon.
• Hybridisation: PCR product is denatured and
then added to a ‘well’ containing the nylon
membrane with the bound probes and
incubated with hybridisation buffer . PCR
product hybridises to probes with
complementary sequences.
• Excess product is washed away during a
series of wash steps.
• Temperature is VERY important during these
stages.

33. Molecular typing Contd…….
• Visualisation of results is achieved by
incubating with a conjugate and enzyme
often streptavidin and horse radish
peroxidase which binds to the biotin of the
PCR product and then adding a substrate.
Band with PCR product turn blue.
• Strips will have internal control bands to
show the test has worked.
• Interpretation is usually achieved by entering
the band pattern into a computer
programme.
• This is an excellent method for low
resolution batch testing.

34. Molecular typing Contd…….
• Sequence Based Typing (SBT)
• DNA sequencing is the determination of
the sequence of a gene and thus is the
highest resolution possible. Sequence
based typing involves PCR amplification of
the gene of interest eg HLA DRB1 followed
by determination of the base sequence.
The sequence is then compared with a
database of DRB1 gene sequences to find
comparable sequences and assign alleles.
This method also allows for detection on
new alleles.

35. Molecular typing Contd…….
• Other molecular methods:
• Reference Strand Conformational Analysis
(RSCA) Offers sequence level typing
without the need to sequence. Assigns
HLA type on the basis of accurate
measurement of conformation i.e. shape
dependent on DNA mobility in
Polyacrylamide gel electrophoresis
(PAGE). Complex and difficult technique
not taken up by labs for routine use.
• Luminex technology – SSOP based. Just
beginning to be introduced into
laboratories for routine use on non urgent
samples.

36. Pros and cons
• Pros:
• Does not require viable cells
• Samples do not have to arrive in the lab the
day they are taken
• PCR SSOP good for batch testing
• Can be semi automated
• Cons:
• Requires good quality DNA
• Require a degree of redundancy within the
primers used
• Sequence of alleles must be known.